#Introduction I am analyzing neogene expression in PDX data.
#Load libraries
library(Seurat)
library(scater)
library(readxl)
library(stringr)
library(tidyr)
library(plyr)
source('../../Utilities/Utilities.r')
source('../../Utilities/Processing_Utilities.r')
#Loading and Processing Data ##importing data from file storage
Files are stored on my MD Anderson drive. I am extracting the the path to each library and generating some meta data.
list.dirs('/Volumes/ludwig_lab/CellRanger/data_output/', recursive = F)
list_of_files <- path_input('/Volumes/ludwig_lab/CellRanger/data_output/', library.split = 7, sample.split = 1)
list_of_files <- list_of_files[-c(1,2,3,5,7,10,12),]
##converting from H5 to seurat objects
PDX_neogenes.list.h5 <- lapply(list_of_files$h5, Read10X_h5)
names(PDX_neogenes.list.h5) <- list_of_files$library_id
PDX_neogenes.list <- lapply( PDX_neogenes.list.h5 ,function(x) {CreateSeuratObject(counts = x) })
names(PDX_neogenes.list) <- list_of_files$library_id
for(i in 1:length(PDX_neogenes.list)) {
PDX_neogenes.list[[i]]<- RenameCells(object = PDX_neogenes.list[[i]], new.names = paste0(
sapply(strsplit(as.character(colnames(PDX_neogenes.list[[i]])), split="-"), "[[", 1),
"-", i))
}
PDX_neogenes <- Reduce(merge, PDX_neogenes.list)
##adding the metadata
PDX_neogenes <- PercentageFeatureSet( PDX_neogenes, pattern = "^MT-", col.name = "percent.mt", assay = 'RNA')
gemgroup <- sapply(strsplit(rownames(PDX_neogenes@meta.data), split="-"), "[[", 2)
PDX_neogenes<- AddMetaData(object=PDX_neogenes, metadata=data.frame(gemgroup=gemgroup, row.names=rownames(PDX_neogenes@meta.data)))
PDX_neogenes$orig.ident <- mapvalues(PDX_neogenes$gemgroup,unique(PDX_neogenes$gemgroup), as.character(list_of_files$library_id))
PDX_neogenes$sample_type <- mapvalues(PDX_neogenes$gemgroup, unique(PDX_neogenes$gemgroup), as.character(list_of_files$sample_type))
PDX_labels <- read.csv('../PDX_dissociation_labels.csv')
PDX_labels
PDX_neogenes$lab_id <- mapvalues(PDX_neogenes$orig.ident, unique(PDX_neogenes$orig.ident), c('DSRCT-1', 'DSRCT-4', 'DSRCT-2',
'ES-1','CDS-1', 'ES-4',
'ES-6', 'OS-SA98', 'OS-31',
'OS-1'))
PDX_neogenes@meta.data %>% group_by(orig.ident, lab_id) %>% summarise(n=n())
##quality control
RidgePlot(PDX_neogenes, features = 'nCount_RNA', group.by = 'lab_id')
RidgePlot(PDX_neogenes, features = 'nFeature_RNA', group.by = 'lab_id')
RidgePlot(PDX_neogenes, features = 'percent.mt', group.by = 'lab_id')
PDX_neogenes
##Outlier Based on OSCA
PDX_neogenes@active.ident <- factor(PDX_neogenes$lab_id)
PDX_neogenes.sce <- as.SingleCellExperiment(PDX_neogenes)
discard.mito <- isOutlier(PDX_neogenes.sce$percent.mt,
type="higher", batch=PDX_neogenes.sce$lab_id, nmads = 2)
mito.plot <- plotColData(PDX_neogenes.sce, x="lab_id", y="percent.mt",
colour_by=I(discard.mito)) + theme(axis.text.x = element_text(angle =90)) + xlab('') + labs(fill = 'Outlier') + ylab('% Mitochondrial Mapping')
discard.counts <- isOutlier(PDX_neogenes.sce$nCount_RNA, nmads = 2, log = TRUE,
type="lower", batch=PDX_neogenes.sce$lab_id)
counts.plot <- plotColData(PDX_neogenes.sce, x="lab_id", y="nCount_RNA",
colour_by=I(discard.counts)) + theme(axis.text.x = element_text(angle =90)) + xlab('') + ylab('Counts')
discard.features <- isOutlier(PDX_neogenes.sce$nFeature_RNA, nmads = 2, log = TRUE,
type="lower", batch=PDX_neogenes.sce$lab_id)
gene.plot <- plotColData(PDX_neogenes.sce, x="lab_id", y="nFeature_RNA",
colour_by=I(discard.features)) + theme(axis.text.x = element_text(angle =90)) + xlab('') + ylab('Genes')
mito.plot
counts.plot + scale_y_log10()
gene.plot + scale_y_log10()
##save original data
saveRDS(PDX_neogenes, '/Volumes/ludwig_lab/NeoGenes/RDS/PDX_neogenes_original.rds')
#Processed data ##filter out low quality cells
PDX_neogenes <- AddMetaData(PDX_neogenes, discard.counts, col.name = 'discard.counts')
PDX_neogenes <- AddMetaData(PDX_neogenes, discard.mito, col.name = 'discard.mito')
PDX_neogenes <- AddMetaData(PDX_neogenes, discard.features, col.name = 'discard.features')
PDX_neogenes_filtered <- subset(PDX_neogenes, subset = discard.features == FALSE &
#discard.mito == FALSE &
discard.counts == FALSE &
percent.mt < 10 & nCount_RNA > 1000 & nFeature_RNA > 500)
VlnPlot(PDX_neogenes, features = 'WT1')
##quality control
RidgePlot(PDX_neogenes_filtered, features = 'nCount_RNA', group.by = 'lab_id')
RidgePlot(PDX_neogenes_filtered, features = 'nFeature_RNA', group.by = 'lab_id')
RidgePlot(PDX_neogenes_filtered, features = 'percent.mt', group.by = 'lab_id')
PDX_neogenes_filtered
##normalize
PDX_neogenes_filtered <- PDX_neogenes_filtered %>%
NormalizeData() %>%
FindVariableFeatures() %>%
ScaleData() %>%
RunPCA()
ElbowPlot(PDX_neogenes_filtered)
PDX_neogenes_filtered <- RunUMAP(PDX_neogenes_filtered, dims = 1:15)
saveRDS(PDX_neogenes_filtered, '/Volumes/ludwig_lab/NeoGenes/RDS/2023-05-1 PDX_neogenes_filtered.rds')
#Load Processed Data
PDX_neogenes_filtered <- readRDS( '/Volumes/ludwig_lab/NeoGenes/RDS/2023-05-1 PDX_neogenes_filtered.rds')
Registered S3 method overwritten by 'data.table':
method from
print.data.table
Registered S3 method overwritten by 'htmlwidgets':
method from
print.htmlwidget tools:rstudio
PDX_neogenes_filtered@active.ident <- factor(PDX_neogenes_filtered$lab_id)
DimPlot(PDX_neogenes_filtered, group.by = 'lab_id', label = T)
FeaturePlot(PDX_neogenes_filtered, 'COL1A1')
FeaturePlot(PDX_neogenes_filtered, 'AR')
FeaturePlot(PDX_neogenes_filtered, 'CCND1')
#Generate Signatures The gene signatures are used to confirm the phenotype of the various sarcoma.
VlnPlot(PDX_neogenes_filtered, features = names(rename_vector))
VlnPlot(PDX_neogenes_filtered, features = 'TP53')
VlnPlot(PDX_neogenes_filtered, features = 'NFATC2')
VlnPlot(PDX_neogenes_filtered, features = 'EWS.FLI1_Signature') + NoLegend()
VlnPlot(PDX_neogenes_filtered, features = 'Ew-NG8') + NoLegend()
FeaturePlot(PDX_neogenes_filtered, 'AR_Signature')
#Neogene analysis Use grep to pull DSRCT-neogenes from the gene list and observe DSRCT-neogene expression
dsrct_ng <- rownames(PDX_neogenes_filtered)[grep('DSRCT', rownames(PDX_neogenes_filtered))]
DotPlot(PDX_neogenes_filtered, features = dsrct_ng, group.by = 'lab_id') + theme(axis.text.x = element_text(angle = 45, hjust = 1, size = 10)) + labs(x='',y ='')
genes <- rownames(PDX_neogenes_filtered)[grep('DSRCT-NG', rownames(PDX_neogenes_filtered))]
DotPlot(PDX_neogenes_filtered, features = genes) + theme(axis.text.x = element_text(angle =45, hjust=1))
Generate a DSRCT_neoegene signature
PDX_neogenes_filtered@meta.data <- PDX_neogenes_filtered@meta.data %>% dplyr::rename(Cluster1 = 'DSRCT_NG')
Error in `dplyr::rename()`:
! Can't rename columns that don't exist.
✖ Column `DSRCT_NG` doesn't exist.
Backtrace:
1. PDX_neogenes_filtered@meta.data %>% dplyr::rename(Cluster1 = "DSRCT_NG")
3. dplyr:::rename.data.frame(., Cluster1 = "DSRCT_NG")
Compare the DSRCT_neogene signature to the EWS.WT1_signature
VlnPlot(PDX_neogenes_filtered, 'DSRCT_NG') + NoLegend()
VlnPlot(PDX_neogenes_filtered, 'EWS.WT1_Signature') + NoLegend()
#Heatmap
##Perform correlation Find genes that correlate with neogenes
cells <- grep('DSRCT',PDX_neogenes_filtered$lab_id)
DSRCT_NG_cor_vector <- apply(PDX_neogenes_filtered@assays$RNA@data[,cells],
1, function(x) cor(x,PDX_neogenes_filtered$DSRCT_NG[cells]))
DSRCT_NG_cor_vector_df <- data.frame(r2=DSRCT_NG_cor_vector, genes = names(DSRCT_NG_cor_vector))
genes <- DSRCT_NG_cor_vector_df %>% top_n(25, r2) %>% pull(genes)
DotPlot(PDX_neogenes_filtered, features = genes) + theme(axis.text.x = element_text(angle =45, hjust=1))
genes <- DSRCT_NG_cor_vector_df %>% top_n(25, r2) %>% pull(genes)
DotPlot(PDX_neogenes_filtered, features = genes) + theme(axis.text.x = element_text(angle =45, hjust=1))
genes <- DSRCT_NG_cor_vector_df %>% top_n(25, r2) %>% pull(genes)
DotPlot(PDX_neogenes_filtered, features = genes) + theme(axis.text.x = element_text(angle =45, hjust=1))
genes <- DSRCT_NG_cor_vector_df %>% top_n(30, r2) %>% pull(genes)
DotPlot(PDX_neogenes_filtered, features = genes) + theme(axis.text.x = element_text(angle =45, hjust=1))
genes <- DSRCT_NG_cor_vector_all_df %>% top_n(30, r2) %>% pull(genes)
DotPlot(PDX_neogenes_filtered, features = genes) + theme(axis.text.x = element_text(angle =45, hjust=1))
genes <- DSRCT_NG_cor_vector_all_df[-grep('DSRCT', DSRCT_NG_cor_vector_all_df$genes),] %>% top_n(30, r2) %>% pull(genes)
DotPlot(PDX_neogenes_filtered, features = genes) + theme(axis.text.x = element_text(angle =45, hjust=1))
DSRCT_NG_cor_vector_all <- apply(PDX_neogenes_filtered@assays$RNA@data[,],
1, function(x) cor(x,PDX_neogenes_filtered$DSRCT_NG[]))
DSRCT_NG_cor_vector_all_df <- data.frame(r2=DSRCT_NG_cor_vector_all, genes = names(DSRCT_NG_cor_vector_all))
length(stri_remove_empty(gene_sigs$EWS.WT1))
[1] 67
#use AddModuleScore to assess the signature expression in each library
PDX_neogenes_filtered <- AddModuleScore(PDX_neogenes_filtered, features = list(DSRCT_gene_signature), name = 'Cluster', ctrl = 50)
#PDX_neogenes_filtered@meta.data <- PDX_neogenes_filtered@meta.data %>% dplyr::select(-c(names(rename_vector)))
PDX_neogenes_filtered@meta.data <- PDX_neogenes_filtered@meta.data %>% dplyr::rename('DSRCT_ng_correlates' = 'Cluster1')
##Compare correlates to EWS.WT1_signature
VlnPlot(PDX_neogenes_filtered, 'DSRCT_ng_correlates') + NoLegend()
VlnPlot(PDX_neogenes_filtered, 'EWS.WT1_Signature')+ NoLegend()
###split data into testing and training groups and find scores
set.seed(123)
split_data <- function(seurat_object, gene_sets){
groups <- sample(c("test", "training"), size = dim(seurat_object)[2], replace = TRUE)
names(groups) <- colnames(seurat_object)
seurat_object <- AddMetaData(object = seurat_object, metadata = groups, col.name = "group")
seurat_object@active.assay <- 'RNA'
obj.list <- SplitObject(seurat_object, split.by = "group")
for (i in 1:length(gene_sets)){
obj.list <- map(obj.list,
AddModuleScore,
features = list(gene_sets[[i]]),
name = names(gene_sets)[i])
}
return(obj.list)
}
roc_plot <- function(test_df,
bias_score = 'length_bias_score',
random_score = 'random_score',
bias_label = 'Length Bias'){
require(pROC)
# Plot ROC curv
roc_bias <- roc(test_df$class, test_df$probabilities_bias)
roc_random <- roc(test_df$class, test_df$probabilities_random)
auroc <- c(auc(roc_bias)[1],
auc(roc_random)[1])
roc_ggplot <- ggroc(list(roc_bias,roc_random)) +
labs(x = "False Positive Rate (FPR)", y = "True Positive Rate (TPR)", color = "") +
scale_color_manual(values = c("limegreen", "darkgray"),
labels = c(paste0(bias_label, "\nScore ", '(AUC = ', round(auroc, 2)[1], ')'),
paste0("Random ", '(AUC = ', round(auroc, 2)[2], ')'))) +
theme_classic() +
theme(legend.position = c(0.7, 0.25),
legend.background = element_rect(fill = NA))
return(roc_ggplot)
}
add_module_scores <- function(seurat_object,
EWS.WT1_score = "",
NG_correlate_score = ""){
gene_sets <- list("EWS.WT1_score" = EWS.WT1_score,
"NG_correlate_score" = NG_correlate_score,
"random_score_ng" = sample(rownames(seurat_object),
size = length(NG_correlate_score), replace = F),
"random_score_ews_wt1" = sample(rownames(seurat_object),
size = length(EWS.WT1_score), replace = F)
)
obj.list <- split_data(seurat_object, gene_sets)
return(obj.list)
}
#Length Bias score We will divide the library to a training set and a test set. First, let us find the signature in the training set:
obj.list <- add_module_scores(PDX_neogenes_filtered,
gene_sigs$EWS.WT1,
DSRCT_gene_signature)
Error in map(obj.list, AddModuleScore, features = list(gene_sets[[i]]), :
could not find function "map"
obj.list <- add_module_scores(PDX_neogenes_filtered,
gene_sigs$EWS.WT1,
DSRCT_gene_signature)
Warning: The following features are not present in the object: , not searching for symbol synonymsWarning: The following features are not present in the object: , not searching for symbol synonyms
EWS.WT1__logistic_random <- glm(class ~ random_score, train_df, family = "binomial")
Error: unexpected symbol in:
"EWS.WT1__logistic_random <- glm(clas
EWS.WT1__logistic_random"
#train model on ES data
train_df <- data.frame(
EWS.WT1_score = obj.list$training$EWS.WT1_score1,
random_score = obj.list$training$random_score_ews_wt11,
class = factor(ifelse(obj.list$training$sample_type == "DSRCT", 1, 0))
)
EWS.WT1_logistic_fit <- glm(class ~ EWS.WT1_score, train_df, family = "binomial")
#summary(logistic_fit)
EWS.WT1_logistic_random <- glm(class ~ random_score, train_df, family = "binomial")
#summary(logistic_random)
train_df <- data.frame(
NG_correlate_score = obj.list$training$NG_correlate_score1,
random_score = obj.list$training$random_score_ng1,
class = factor(ifelse(obj.list$training$sample_type == "DSRCT", 1, 0))
)
NG_correlate_logistic_fit <- glm(class ~ NG_correlate_score, train_df, family = "binomial")
NG_correlate_logistic_random <- glm(class ~ random_score, train_df, family = "binomial")
#summary(logistic_fit)
# Predict probabilities on test set
test_df <- data.frame(
EWS.WT1_score = obj.list$test$EWS.WT1_score1,
random_score = obj.list$test$random_score_ews_wt11,
class = factor(ifelse(obj.list$test$sample_type == "DSRCT", 1, 0))
)
test_df$probabilities_bias <- predict(EWS.WT1_logistic_fit, newdata = test_df, type = "response")
test_df$probabilities_random <- predict(EWS.WT1_logistic_random, newdata = test_df, type = "response")
plot(probabilities_bias ~ EWS.WT1_score, data = test_df)
plot(probabilities_bias ~ random_score, data = test_df)
ggplot(test_df, mapping = aes(y = probabilities_bias, x = EWS.WT1_score)) + geom_point() + theme_classic() + labs(x = 'EWS.WT1_score',y= 'Probability of DSRCT')
EWS.WT1_score_plot <- roc_plot(test_df,
bias_score = 'EWS.WT1_score',
random_score = 'random_score',
bias_label = 'EWS.WT1_score')
Setting levels: control = 0, case = 1
Setting direction: controls < cases
Setting levels: control = 0, case = 1
Setting direction: controls > cases
EWS.WT1_score_plot
# Predict probabilities on test set
test_df <- data.frame(
NG_correlate_score = obj.list$test$NG_correlate_score1,
random_score = obj.list$test$random_score_ng1,
class = factor(ifelse(obj.list$test$sample_type == "DSRCT", 1, 0))
)
test_df$probabilities_bias <- predict(NG_correlate_logistic_fit, newdata = test_df, type = "response")
test_df$probabilities_random <- predict(NG_correlate_logistic_random, newdata = test_df, type = "response")
plot(probabilities_bias ~ NG_correlate_score, data = test_df)
plot(probabilities_bias ~ random_score, data = test_df)
ggplot(test_df, mapping = aes(y = probabilities_bias, x = NG_correlate_score)) + geom_point() + theme_classic() + labs(x = 'EWS.WT1_score',y= 'Probability of DSRCT')
NG_correlate_score_plot <- roc_plot(test_df,
bias_score = 'NG_correlate_score',
random_score = 'random_score',
bias_label = 'NG_correlate_score')
Setting levels: control = 0, case = 1
Setting direction: controls < cases
Setting levels: control = 0, case = 1
Setting direction: controls < cases
NG_correlate_score_plot
#find genes for each DSRCT
DSRCT_NG_cor_vector_list <- lapply(c("DSRCT-1","DSRCT-4","DSRCT-2"), function(x) {
cells <- which(PDX_neogenes_filtered$lab_id == x)
cor_vector <- apply(PDX_neogenes_filtered@assays$RNA@data[,cells],
1, function(x) cor(x,PDX_neogenes_filtered$DSRCT_NG[cells]))
cor_vector_df <- data.frame(r2=cor_vector, genes = names(cor_vector))
cor_vector_df
}
Error: Incomplete expression: DSRCT_NG_cor_vector_list <- lapply(c("DSRCT-1","DSRCT-4","DSRCT-2"), function(x) {
cells <- which(PDX_neogenes_filtered$lab_id == x)
cor_vector <- apply(PDX_neogenes_filtered@assays$RNA@data[,cells],
1, function(x) cor(x,PDX_neogenes_filtered$DSRCT_NG[cells]))
cor_vector_df <- data.frame(r2=cor_vector, genes = names(cor_vector))
cor_vector_df
}
DSRCT_NG_cor_vector_list <- lapply(c("DSRCT-1","DSRCT-4","DSRCT-2"), function(x) {
cells <- which(PDX_neogenes_filtered$lab_id == x)
cor_vector <- apply(PDX_neogenes_filtered@assays$RNA@data[,cells],
1, function(x) cor(x,PDX_neogenes_filtered$DSRCT_NG[cells]))
cor_vector_df <- data.frame(r2=cor_vector, genes = names(cor_vector))
cor_vector_df
})
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##dotplots for each PDX
genes <- DSRCT_NG_cor_vector_list[['DSRCT-2']][-grep('DSRCT', DSRCT_NG_cor_vector_list[['DSRCT-2']]$genes),] %>% top_n(30, r2) %>% pull(genes)
DotPlot(PDX_neogenes_filtered, features = genes) + theme(axis.text.x = element_text(angle =45, hjust=1))
genes <- DSRCT_NG_cor_vector_list[['DSRCT-4']][-grep('DSRCT', DSRCT_NG_cor_vector_list[['DSRCT-4']]$genes),] %>% top_n(30, r2) %>% pull(genes)
DotPlot(PDX_neogenes_filtered, features = genes) + theme(axis.text.x = element_text(angle =45, hjust=1))
genes <- rownames(PDX_neogenes_filtered)[grep('Ew-NG', rownames(PDX_neogenes_filtered))]
DotPlot(PDX_neogenes_filtered, features = genes) + theme(axis.text.x = element_text(angle =45, hjust=1))
genes <- rownames(PDX_neogenes_filtered)[grep('CIC-NG', rownames(PDX_neogenes_filtered))]
DotPlot(PDX_neogenes_filtered, features = genes) + theme(axis.text.x = element_text(angle =45, hjust=1))
DEGs <- FindAllMarkers(PDX_neogenes_filtered, only.pos = T, test.use = 'LR', return.thresh = 0.05, max.cells.per.ident = 1000)
top_DEGs <- DEGs %>% group_by(cluster) %>% top_n(20, avg_log2FC)
top_DEGs <- DEGs %>% group_by(cluster) %>% top_n(3, avg_log2FC) %>% pull(gene)
DotPlot(PDX_neogenes_filtered, features = top_DEGs) + theme(axis.text.x = element_text(angle =45, hjust=1))
DotPlot(PDX_neogenes_filtered, features = c('CIC.DUX4_Signature', 'EWS.WT1_Signature', 'EWS.FLI1_Signature')) + theme(axis.text.x = element_text(angle =45, hjust=1)) + labs(x='', y ='')
Why does OS31 have NFATC genes
VlnPlot(PDX_neogenes_filtered, features = 'CIC')
VlnPlot(PDX_neogenes_filtered, features = 'NFATC2')
VlnPlot(PDX_neogenes_filtered, features = 'NFAT-NG41')
genes <- rownames(PDX_neogenes_filtered)[grep('NFAT-NG', rownames(PDX_neogenes_filtered))]
DotPlot(PDX_neogenes_filtered, features = genes) + theme(axis.text.x = element_text(angle =45, hjust=1))